The accumulation of alpha-synuclein (ASYN)-containing Lewy bodies and associated degeneration of dopamine (DA) neurons are major pathological hallmarks of Parkinson's disease (PD). The autophagy- lysosome pathway (ALP) is an important intracellular degradation and recycling pathway and a compromise in ALP function promotes the accumulation of toxic ASYN species. As the ALP is altered in PD, identification and validation of novel ALP targets which promote ASYN clearance is a timely approach in the development of novel PD therapeutics. ATP6V0C forms the proton translocating channel of V-ATPase, an enzyme complex that regulates lysosomal acidification and clearance of ALP substrates. High concentrations of bafilomycin, a macrolide antibiotic that binds ATP6V0C with high affinity, inhibits V-ATPase and produces neurotoxicity related to its inhibition of lysosome function and ASYN clearance. However, concentrations of bafilomycin too low to inhibit V-ATPase attenuate cell death and endogenous ASYN accumulation resulting from lysosome dysfunction, and inhibit DA neuron death resulting from ASYN over-expression in vivo. While bafilomycin has a narrow therapeutic index which limits its long-term use in human PD patients, our data point to ATP6V0C as a novel target for promoting ASYN clearance and cell survival. However, whether ATP6V0C mediates these protective effects of bafilomycin and if over-expression of ATP6V0C itself can provide neuroprotective benefit in the absence of bafilomycin has not been tested. We hypothesize that ATP6V0C mediates the bafilomycin- dependent clearance of toxic ASYN species and attenuates ASYN-associated neurotoxicity. In Aim 1, we will genetically over-express ATP6V0C in cultured mammalian cells to determine its relative importance in regulating the clearance of endogenous ASYN, ALP function and cell death. We will also determine if the over-expression of human ATP6V0C in mouse substantia nigra (SN) attenuates ASYN accumulation and associated neurodegeneration resulting from over-expression of wild-type human ASYN. In Aim 2, we will genetically knockdown ATP6V0C or introduce point mutations in ATP6V0C that confer resistance to bafilomycin-mediated inhibition of V-ATPase, to determine the requirement of ATP6V0C in regulating such neuroprotective functions of bafilomycin in cultured mammalian cells. We will also develop bafilomycin analogs with reduced V-ATPase inhibitory activity to determine if bafilomycin mediated neuroprotection is enhanced with a reduction in V-ATPase inhibitory activity. These studies will validate the utility of ATP6V0C as a novel ALP-dependent target for the future generation of PD therapeutics which promotes ASYN clearance and DA neuron survival. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is a leading age-related neurodegenerative disease and our laboratory studies the mechanisms by which toxic alpha synuclein species accumulate in PD and contribute to PD neuropathology and neurodegeneration. Specifically, our laboratory focuses on how intracellular pathways that regulate protein degradation may negatively influence DA neuron survival in PD. For this application we hypothesize that the protein ATP6V0C, a subunit of the vacuolar ATPase (V-ATPase) complex of lysosomes and other acidic organelles, is a valid target for PD therapeutics and that its over-expression or pharmacological regulation may promote DA neuron survival through its ability to increase the clearance of toxic alpha synuclein species from cells. In summary, our research will validate whether over-expression of ATP6V0C is an effective strategy for promoting alpha synuclein clearance, and if pharmacological regulation of this target also affords similar neuroprotection.